Of the existing cytotoxic active ingredients for treating tumours, Taxol.RTM. (Paclitaxel; Bristol-Myers Squibb), a microtubuli-stabilising agent, plays an important role and has remarkable commercial success. However, Taxol has a number of disadvantages. In particular, its very poor solubility in water is a problem. It therefore became necessary to administer Taxol.RTM. in a formulation with Cremophor EL.RTM. (polyoxyethylated castor oil; BASF, Ludwigshafen, Germany). Cremophor EL.RTM. has severe side effects; for example it causes allergies which in at least one case have led even to the death of a patient.
Although the Taxan class of microtubuli-stabilising anti-cancer agents has been commended as "perhaps the most important addition to the pharmaceutical armoury against cancer in the last decade" (see Rowinsky E. K., Ann. rev. Med. 48, 353-374 (1997)), and despite the commercial success of Taxol.RTM., these compounds still do not appear to represent a really great breakthrough in the chemotherapy of cancer. Treatment with Taxol.RTM. is linked with a series of significant side effects, and a few primary classes of compact tumours, namely colon and prostate tumours, respond to this compound only to a small extent (see Rowinsky E. K., inter alia). In addition, the efficacy of Taxol can be impaired and even completely neutralised by acquired resistance mechanisms, especially those based on the overexpression of phosphoproteins, which act as efflux pumps for active ingredients, such as "Multidrug Resistance" due to overexpression of the multidrug transport glycoprotein "P-glycoprotein".
Epothilones A and B represent a new class of microtubuli-stabilising cytotoxic active ingredients (see Gerth, K. et al., J. Antibiot. 49, 560-3 (1966)) of the formulae: ##STR1##
wherein R signifies hydrogen (epothilone A) or methyl (epothilone B).
These compounds have the following advantages over Taxol.RTM.:
a) They have better water-solubility and are thus more easily accessible for formulations. PA1 b) It has been reported that, in cell culture experiments, they are also active against the proliferation of cells, which, owing to the activity of the P-glycoprotein efflux pump making them "multidrug resistant", show resistance to treatment with other chemotherapy agents including Taxol.RTM. (see Bolag, D. M., et al., "Epothilones, a new class of microtubuli-stabilizing agents with a Taxol-like mechanism of action", Cancer Research 55, 2325-33 (1995)). And PA1 c) it could be shown that they are still very effective in vitro against a Taxol.RTM.-resistant ovarian carcinoma cell line with modified .beta.-tubulin (see Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541 (1997)). PA1 molecular filtration (gel chromatography), e.g. on a column of material such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol as eluant; PA1 separation of the epothilones by reversed-phase chromatography after being taken up in a suitable solvent, and elution with a mixture of nitrile/water (compulsory), preferably characterised in that the chromatography is carried out on a column of material, especially a RP-18 material, which is charged with hydrocarbon chains, such as hydrocarbon chains containing 18 carbon atoms, and an eluant comprising a nitrile, especially a lower alkyl-nitrile, in particular acetonitrile, is used, in particular a mixture of nitrile/water is used, especially a mixture of acetonitrile/water, preferably in a ratio of nitrile to water of about 1:99 to 99:1, primarily between 1:9 and 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6. PA1 single or multiple extraction of the residue (especially after evaporation) in a two-phase system consisting of water and a solvent immiscible with water, preferably an ester, in particular a lower alkyl lower alkanoate, such as ethyl acetate or isopropyl acetate; PA1 adsorption chromatography, in particular by adding to a column of silica gel and eluting with an appropriate solvent or solvent mixture, especially a mixture of ester/hydrocarbon, for example lower alkyl alkanoate/C.sub.4 -C.sub.10 -alkane, especially ethyl or isopropyl acetate/n-hexane, in which the ratio between the ester and hydrocarbon is preferably in the range 99:1 to 1:99, preferably 10:1 to 1:10, for example 4:1; PA1 dissolving the residue, which may be obtained after concentration, in an appropriate solvent such as an alcohol, e.g. methanol; PA1 mixing with activated carbon and removal thereof; PA1 recrystallisation, e.g. from appropriate solvents or solvent mixtures, for example consisting of esters, ester/hydrocarbon mixtures or alcohols, especially ethyl or isopropyl acetate: toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothilone B); PA1 either by direct separation of the epothilones from one another by reversed-phase chromatography after being taken up in an appropriate solvent, for example a nitrile/water mixture, especially an acetonitrile/water mixture (ratio of nitrile to water 1:99 to 99:1, preferably 1:9 to 9:1, especially 3:1), if necessary after filtration or centrifugation, preferably on a silica gel that has been derivatized by hydrocarbon radicals, e.g. a silica gel modified by alkyl radicals containing 8 to 20, especially 18, C-atoms, eluting with an eluant comprising a nitrile, especially a lower alkylnitrile, such as acetonitrile, especially a mixture of the nitrile with water, such as an acetonitrile/water mixture, whereby detection of the interesting fractions is effected in conventional manner, for example by UV detection or (preferably) by on-line HPLC (HPLC with a very small column, the analyses taking less than 1 minute, and detection e.g. at 250 nm), this enabling a particularly exact separation of the fractions containing the desired product to take place; if required, with subsequent concentration, for example by distillation, to remove the nitrile; if desired, with subsequent single or multiple, for example double, extraction of the residue of evaporation in a two-phase system consisting of water and an immiscible solvent, such as ethyl acetate or isopropyl acetate; additional concentration of the organic phase and dissolving of the residue in an appropriate solvent, preferably an ester such as ethyl acetate or isopropyl acetate, if required, filtration or centrifugation, if desired adding to a column of silica gel and eluting with an appropriate solvent or solvent mixture, for example with a mixture of ester/hydrocarbon, e.g. lower alkyl alkanoate/C.sub.4 -C.sub.10 -alkane, especially ethyl or isopropyl acetate/n-hexane, in which the ratio of ester to hydrocarbon is preferably in the range 99:1 to 1:99, preferably 10:1 to 1:10, e.g. 4:1; subsequent combining of the fractions containing each desired epothilone, especially epothilone A or epothilone B, and after removing the solvent, for example by distillation, preferably concentrating to dryness; then, dissolving of the residue in an appropriate alcohol, preferably methanol; and if desired, in order to obtain especially high purity, mixing with activated carbon and then separating the activated carbon, for example by filtration; and finally, by recrystallisation as described below under variant 2 (for epothilone B in particular from methanol), separate extraction of the epothilones, especially epothilones A or B. This is the most preferred variant 1, the outstanding characteristic of which is the surprising direct separation by reversed-phase chromatography of the epothilone-containing mixture desorbed by the resin, despite all the impurities in the organic extract; PA1 or (variant 2) first of all exclusion chromatography takes place (molecular filtration) e.g. on a column of material such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol as eluant, and then subsequent separation of the epothilones present in the peak fractions obtained, e.g. epothilone A and B, by reversed-phase chromatography as described above for variant 1; if required twice, if peak fractions of one epothilone contain those of another, for example if those with epothilone A still contain residues of epothilone B; and then separate recrystallisation of each epothilone from appropriate solvents or solvent mixtures, for example from ethyl or isopropyl acetate:toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothilone B). This is variant 2 of working up and purification. PA1 R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H, --O--(Alk(--R)--O--).sub.p --H or PA1 --O--(alk(--R)--O--).sub.q --alk--CO--Y; alk in all cases is alkyl, especially lower alkyl; m, n, p, q and z are a whole number from 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR.sub.1 or NR.sub.2 R.sub.3, wherein R.sub.1, R.sub.2 and R.sub.3 independently of one another, are hydrogen or lower alkyl, or R.sub.2 and R.sub.3 combined together with the linking nitrogen signify morpholino, piperidino, pyrrolidino or piperazino; PA1 or a branched cyclodextrin, in which etherifications or acetals with other sugars are present, and which are selected from glucosyl-, diglucosyl-(G.sub.2 -.beta.-cyclodextrin), maltosyl- or di-maltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- and galactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl ester of a cyclodextrin; and PA1 b) comprises a step for separating the epothilones, especially epothilones A and B, from one another, which is characterised by chromatography on a reversed-phase column with an eluant containing a lower alkylcyanide, the chromatography being carried out on a column material, especially an RP-18 material, which is charged with hydrocarbon chains containing 18 carbon atoms, and employing an eluant containing a lower alkyinitrile, especially acetonitrile, in particular a mixture of lower alkylnitrile/water, preferably a mixture of acetonitrile/water, preferably in a ratio of lower alkylnitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6, whereby if desired, it is possible to use further steps for working up and purification.
Pharmaceutical application of the epothilones, for example for tumour treatment, is possible in an analogous manner to that described for Taxol, see for example U.S. Pat. No. 5,641,803; U.S. Pat. No. 5,496,804; U.S. Pat. No. 5,565,478).
In order to be able to use the epothilones on a larger scale for pharmaceutical purposes, however, it is necessary to obtain appropriate amounts of these compounds.
Until now, the extraction of natural substances by means of myxobacteria, especially the epothilones from the cell strain Sorangium cellulosum Soce90 (deposited under no. 6773 at the German Collection of Microorganisms, see WO 93/10121) was described in literature. In order to obtain a satisfactory concentration of the natural substances, especially the epothilones, in the culture medium for the subsequent extraction, previously an adsorbate resin based on polystyrene was always added, for example Amberlite XAD-1180 (Rohm & Haas, Frankfurt, Germany).
However, the disadvantage of this process is that, on a large scale, it leads to an abundance of problems. Valves are impaired by the globules of resin, pipes can block, and apparatus may be subject to greater wear due to mechanical friction. The globules of resin are porous and therefore have a large inner surface area (about 825 m.sup.2 /gram resin). Sterilisation becomes a problem, as air enclosed in the resin is not autoclaved. Thus, the process cannot be practicably carried out on a large scale using resin addition.
On the other hand, without adding resin globules, a satisfactory concentration of epothilones cannot be achieved in the culture medium.
Surprisingly, the requirements for finding a way out of this dilemma have now been found, enabling a satisfactory concentration of natural substances to be obtained from microorganisms, in particular myxobacteria, which produce epothilones such as epothilone A or B, in particular a concentration of epothilones A and B, in the culture medium, without the addition of resins, and thus enabling production of these compounds, especially epothilones to be carried out on a technical and industrial scale without the above-mentioned disadvantages.